Three-Dimensional Thymic Culture System to Generate iPSC-Derived Thymic Emigrants

Published: July 31, 2023

Abstract

Source: Vizcardo, R., et al. A Three-dimensional Thymic Culture System to Generate Murine Induced Pluripotent Stem Cell-derived Tumor Antigen-specific Thymic Emigrants. J. Vis. Exp. (2019).

This video demonstrates a protocol for generating functional antigen-specific T cells from iPSC-derived immature T cells using a three-dimensional thymic culture system. Co-culturing the iPSC-derived immature T cells with endogenous lymphocyte-depleted mice fetal thymic lobes resulted in successful maturation of the T cells to form CD8αβ+ MHC class I+ iPSC-derived thymic emigrants (iTEs).

Protocol

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board. 1. 3D Thymic Organ Culture to Generate iTE Harvest mouse fetal thymic lobes and deploy endogenous lymphocytes by deoxyguanosine (dGUO) treatment. On day 7 of dGUO treatment, take four new 10 cm dishes and fill each with 20 mL of complete media (Roswell Park Memorial Institute Media 1640 [RPMI 1640] + 10% FBS + 1x…

Representative Results

Figure 1: Immunohistochemistry of thymic lobes seeded with iPSC-derived immature T cells. Top: H&E staining of a thymic lobe with and without seeding of iPSC-derived immature T cells. From second top to bottom: confocal images of the sectioned lobes stained with DAPI (nucleus), CD3 (T cell), and merge. Scale bars = 100 µm. <p class="jove_content" fo:keep-together.wit…

Offenlegungen

The authors have nothing to disclose.

Materials

 2-deoxyguanosine Sigma-Aldrich 312693-72-4
2-Mercaptoethanol (1,000x)  Thermo Fisher Scientific  21985-023
FBS  Gemini  100-500
Flt-3 ligand  R&D Systems  427-FL
Interleukin-7  R&D Systems  407-ML
MEM Non-Essential Amino Acids Solution Gibco 11140050
MEM powder Gibco 61100061
Sodium Pyruvate  Thermo Fisher Scientific  11360-070
Stem Cell Factor (SCF)  R&D Systems  455-MC
Penicillin/streptomycin Thermo Fisher Scientific 15140-122
Phosphate-buffered saline pH 7.4 (1x) Thermo Fisher Scientific 10010-023
RPMI 1640 Gibco 11875093
10 cm dish Corning, Inc 353003
Perfecta3D Hanging Drop Plate  Sigma-Aldrich  HDP1096
Forceps DUMONT 0108-5PO
OP9/N-DLL1 Riken Bioresource center Cat# RCB2927; RRID:CVCL_B220
GlutaMAX (100x)  Thermo Fisher Scientific  35050-061

Tags

Play Video

Diesen Artikel zitieren
Three-Dimensional Thymic Culture System to Generate iPSC-Derived Thymic Emigrants. J. Vis. Exp. (Pending Publication), e21533, doi: (2023).

View Video